(1) Summary of the Invention
The present invention relates to an improved method for detecting drug metabolites which are transferred from a mother to a newborn infant during pregnancy. In particular, the method involves the isolation of meconium from a newborn infant, separation of the drug metabolites from the meconium in a single solution and assaying for the metabolites in the solution.
(2) Prior Art
The use of illicit drugs in the United States is widespread. According to a national survey in 1985, an estimated 23 million people were users of illicit drugs (1985 National Health Household Survey on Drug Abuse. Rockville, MD., 1987). Although exact figures are not known, a sizable portion of drug users are women of childbearing age or are pregnant women. Infants born to these drug dependent women have multiple problems. In the neonatal period, their mortality rate is increased as well as morbidity, which includes asphyxia, prematurity, low birth weight, hyaline membrane disease, infections, aspiration pneumonia, congenital malformations, abnormal heart rate and breathing patterns and drug withdrawal (Ostrea, E. M., Chavez, C. J., J. Pediatr. 94:292-295, (1979); Zelson, C., Rubio E., and Wasserman, E., Pediatrics 48:178 (1971)). Long term sequelae are not uncommon and include delays in physical growth and mental development, sudden infant death syndrome, hyperactivity, ocular and neurologic abnormalities and lately, a risk to acquired immunodeficiency disease (Wilson, G S., McCreary, M., Kean, J., and Baxter, J., Pediatrics 63:135-144 (1979); Chavez, C. J., et al., J. Pediatrics 95:407-409 (1979); Chasnoff, I. J., et al., Pediatrics 70:210-213 (1982); Chavez, C. J., et al., Pediatr Res 12:367A, (1979); and Oleske, J., et al., J. Am. Med. Assoc., 249:2345-2349 (1983)). At present, cocaine abuse among pregnant women has also become widespread and infant morbidity, notably cerebrovascular problems have been reported (Chasnoff, I. J., et al., J. Pediatr. 108:456-459 (1986)). Because of these immediate and long term problems, infants of drug dependent women (IDDM) constitute a high risk group and have to be identified as soon as possible after birth if intervention is to be successful.
Unfortunately, the identification of the drug exposed neonate is not easy. Many of the drugs to which the fetus is exposed, in utero, do not produce immediate or recognizable effects in the neonates (Kandall, S. R., Am. J. Dis Child, 127:58-61 (1974)). Maternal admission of drug usage is often inaccurate because of fear of the consequences stemming from such admission. Even with maternal cooperation, such information regarding the type and extent of drug usage is often inaccurate (Ostrea, E. M., et al., J. Pediatr. 88:642-645 (1976)). One alternative is to test the infant's urine for drugs, but this procedure has its limitations since successful detection of drug metabolites in the infant's urine is dependent on time of the last drug intake by the mother or when, after birth, the infant's urine was collected. A high rate of false negative results in neonatal urine tests can arise from the mother's abstention from the use of the drug a few days before she delivers or to the inability to obtain a sample of the infant's urine soon after birth (Halstead, A. C., et al., Clin. Biochem. 21:59-61 (1988)). Our experience verifies this diagnostic problem. Urine from 337 infants of known drug dependent mothers was tested by thin-layer chromatography It was found that only 13% were positive. Similarly, only 37% of the urine samples taken from drug dependent infants were positive for drugs when tested by TDX fluorescent polarization immunoassay (Ostrea, E. M., Brady, M. J., Parks, P. M., Asensio, D. C., Nalaz, A., J. Ped. 115:474-474 (1989)). Even with more sensitive methods such as radioimmunoassay, 8 urine samples tested negative for drugs despite a positive test in the stools (Ostrea, E. M., Brady, M. J., Parks, P. M., Asensio, D. C., Naluz, A., J. Ped. 115:474-474 (1989)). Clearly, there is a need for a better way to detect prenatal drug exposure in this high risk group of infants.
A new method has been developed for identifying fetal drug exposure by detecting drug metabolites in meconium, the first green stools of the newborn infant which is passed within a few days after birth (Ostrea, E. M., Parks, P., Brady, M., Clin. Chem. 34:2372-2373 (1989); Ostrea, E. M., Brady, M. J., Parks, P. M., Asensio, D. C., Naluz, A., J. Ped. 115:474-474 (1989)). The concept behind this method was based on initial research in pregnant, morphine addicted monkeys (Ostrea, E. M., Lynn, S. N., Wayne, R. H., Stryker, J. C., Dev. Pharmacol. Ther. 980; 1:163-170 (1980)) and subsequently in rats (Ostrea, E. M., Brady, M. J., Parks, P. M., Asensio, D. C., Naluz, A., J. Ped. 115:474-477 (1989)) which showed that a high concentration of drug metabolites were present in the gastrointestinal tract of their fetuses We interpreted this observation to be a consequence of the following mechanism: morphine in the fetus is metabolized by the liver to water soluble glucuronide conjugates which are then excreted into the bile or urine (Jaffe, J., Drug addiction and drug abuse. In: Goodman, L., Gillman, A., eds. The Pharmacologic Basis of Therapeutics. London: Collier-Macmillan, 535-584 (1989)). In either case, morphine glucuronide accumulates in the fetal intestines either from bile secretion or from fetal urine which is swallowed via the amniotic fluid Thus, meconium represents an excretion product which is cumulative of the entire gestation. Overall therefore, meconium acts as a reservoir of drug metabolites in the fetus; seemingly a stockpile of pharmacologic waste products throughout gestation.
Clinical studies have been conducted which have validated meconium analysis as a reliable drug screen in the newborn infant:
1. Meconium obtained from 20 infants of drug-dependent mothers and five control infants were analyzed by radioimmunoassay for the metabolites of heroin, cocaine and cannabinoids (Ostrea, E. M., Brady, M. J., Parks, P. M., Asensio, D. C., Naluz, A., J. Ped. 115-474-477 (1989)). Control stools showed no drug. Meconium from the infants of drug-dependent mothers showed the presence of at least one drug metabolite: 80% of the infants of drug-dependent mothers showed cocaine (range 0.14 to 19.91 .mu.g/g stool), 55% showed morphine (range 0.41 to 14.97 .mu.g/g stool), and 60% showed cannabinoid (range 0.05 to 0.67 .mu.g/g stool). The concentrations of metabolites were highest during the first 2 days; some stools tested positive up to the third day. In contrast, only 37% of the infants had positive results on a urine screen (fluorescent polarization immunoassay method).
2. Meconium testing was used to determine the prevalence of illicit drug abuse among pregnant women who delivered in a large perinatal center (Ostrea, E. M., Jr., Brady, M., Gause, S., Raymundo, A. L., Stevens M., Pediat. Res. 27:251A (1990)). A total of 3010 infants were screened for the metabolites of cocaine, morphine and cannabinoids in their meconium by radioimmunoassay: 44.3% were positive for either one of the 3 drug metabolites; 41% were positive for cocaine or morphine, 30.7% were positive for cocaine (15.4% positive for cocaine only); 20.5% positive for morphine (7.3% positive for morphine only) and 11.5% positive for cannabinoid (5.2% positive for cannabinoid only). In contrast, only 11.1% of the mothers in the entire population studied admitted to the use of drugs during pregnancy.
3. The sensitivity of meconium test is high. The method was compared to drug detection by maternal hair analysis and in depth interview of the mother (Ostrea, E. M., Martier, S., Welch, R., Brady, M. Pediatr Res. 27, 219A (1990); Welch, R. A., Martier, S. S., Ager, J. W., Ostrea, E. M., Sokol, R. J., Substance Abuse (in press) (1990)). In 26 high risk mothers studied, the abuse of one drug during pregnancy was identified by history in 19 subjects (73%); by meconium analysis in 19 subjects (73.1%) and by hair analysis in 12/16 (75%) subjects. Abuse of 2 or more drugs was identified only in 6 subjects (23%) by history, as compared to 9 subjects (34.6%) by meconium analysis and in 8 (50%) by hair analysis. There was 96% concordance of cocaine identification in hair and meconium and 73% for heroin and cannabinoids. There was also a high correlation between the cocaine concentration in meconium and in hair.
Meconium is therefore an ideal specimen for drug testing in the newborn period: (i) its collection is easy and non-invasive, (ii) it contains high concentrations of drugs and their metabolites and (ii) drugs may be present in meconium for up to the third day after birth. Meconium testing is sensitive, quantitative, and rapid. The test is therefore useful for diagnostic purposes as well as for clinical and epidemiologic research.
However, the preferred method described in U.S. Pat. application Ser. No. 264,131) is different for mass drug screening of meconium in infants since the former involve separate extractions for the different drugs with acidified water or methanol and analysis by immunoassay, particularly radio-immunoassay. It is desirable to modify the procedure to provide for mass screening and particularly to allow analysis by other methods such as enzyme immunoassay (EMIT), fluorescent polarization method, HPLC and gas chromatography/mass spectroscopy.